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Bim and Mcl-1 exert key roles in regulating JAK2V617F cell survival.

Rubert J, Qian Z, Andraos R, Guthy DA, Radimerski T - BMC Cancer (2011)

Bottom Line: Treatment of JAK2V617F mutant cell lines with a JAK2 inhibitor was found to trigger Bim activation.We conclude that Bim and Mcl-1 have key opposing roles in regulating JAK2V617F cell survival and propose that inactivation of aberrant JAK2 signaling leads to changes in Bim complexes that trigger cell death.Thus, further preclinical evaluation of combinations of JAK2 inhibitors with Bcl-2 family antagonists that also tackle Mcl-1, besides Bcl-xL, is warranted to assess the therapeutic potential for the treatment of chronic myeloproliferative neoplasms.

View Article: PubMed Central - HTML - PubMed

Affiliation: Disease Area Oncology, Novartis Institutes for BioMedical Research, Basel, Switzerland.

ABSTRACT

Background: The JAK2V617F mutation plays a major role in the pathogenesis of myeloproliferative neoplasms and is found in the vast majority of patients suffering from polycythemia vera and in roughly every second patient suffering from essential thrombocythemia or from primary myelofibrosis. The V617F mutation is thought to provide hematopoietic stem cells and myeloid progenitors with a survival and proliferation advantage. It has previously been shown that activated JAK2 promotes cell survival by upregulating the anti-apoptotic STAT5 target gene Bcl-xL. In this study, we have investigated the role of additional apoptotic players, the pro-apoptotic protein Bim as well as the anti-apoptotic protein Mcl-1.

Methods: Pharmacological inhibition of JAK2/STAT5 signaling in JAK2V617F mutant SET-2 and MB-02 cells was used to study effects on signaling, cell proliferation and apoptosis by Western blot analysis, WST-1 proliferation assays and flow cytometry. Cells were transfected with siRNA oligos to deplete candidate pro- and anti-apoptotic proteins. Co-immunoprecipitation assays were performed to assess the impact of JAK2 inhibition on complexes of pro- and anti-apoptotic proteins.

Results: Treatment of JAK2V617F mutant cell lines with a JAK2 inhibitor was found to trigger Bim activation. Furthermore, Bim depletion by RNAi suppressed JAK2 inhibitor-induced cell death. Bim activation following JAK2 inhibition led to enhanced sequestration of Mcl-1, besides Bcl-xL. Importantly, Mcl-1 depletion by RNAi was sufficient to compromise JAK2V617F mutant cell viability and sensitized the cells to JAK2 inhibition.

Conclusions: We conclude that Bim and Mcl-1 have key opposing roles in regulating JAK2V617F cell survival and propose that inactivation of aberrant JAK2 signaling leads to changes in Bim complexes that trigger cell death. Thus, further preclinical evaluation of combinations of JAK2 inhibitors with Bcl-2 family antagonists that also tackle Mcl-1, besides Bcl-xL, is warranted to assess the therapeutic potential for the treatment of chronic myeloproliferative neoplasms.

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Bad and Bim depletion in JAK2V617F mutant SET-2 cells suppress NVP-BSK805-induced apoptosis. A: SET-2 cells were transfected with control siRNA (Ctrl), Bad siRNA or siRNAs to deplete both Bad and Bim simultaneously. After 72 hours, cells were treated with 500 nM NVP-BSK805 or the drug vehicle DMSO for 30 hours and extracted for Western blot analysis. Arrowhead depicts cleaved PARP running below the full-length PARP protein. β-tubulin was probed for as a loading control. B: SET-2 cells were transfected with control siRNA (Ctrl) or Bim siRNA and analyzed as described above. The Bim Western blot depicts an extended molecular weight range to show that Bim-EL is the predominant form of the protein expressed in SET-2 cells. C: SET-2 cells were transfected with control, Bad or Bim siRNAs, or siRNAs to deplete both Bad and Bim simultaneously, for 48 hours and then treated with 1 μM NVP-BSK805 or the drug vehicle DMSO for 40 hours. Controls also consisted of non-transfected cells. Then, DNA content was measured by FACS using propidium iodide (PI) staining and the percentage of cells with less than 2N DNA content was determined (data represent the mean ± SD of three independent experiments). *Significantly different from respective DMSO control using t-test (p < 0.05). #Significant difference between groups as assessed by t-test (p < 0.05). D: SET-2 cells were transfected with control, Bad or Bim siRNAs, or siRNAs to deplete both Bad and Bim simultaneously, for 24 hours and then treated with increasing concentrations of NVP-BSK805 in cell proliferation assays for 48 hours. The histogram depicts half-maximal growth-inhibitory (GI50) concentrations of NVP-BSK805 for each condition (data represent the mean ± SD of two independent experiments carried out in triplicate).
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Figure 3: Bad and Bim depletion in JAK2V617F mutant SET-2 cells suppress NVP-BSK805-induced apoptosis. A: SET-2 cells were transfected with control siRNA (Ctrl), Bad siRNA or siRNAs to deplete both Bad and Bim simultaneously. After 72 hours, cells were treated with 500 nM NVP-BSK805 or the drug vehicle DMSO for 30 hours and extracted for Western blot analysis. Arrowhead depicts cleaved PARP running below the full-length PARP protein. β-tubulin was probed for as a loading control. B: SET-2 cells were transfected with control siRNA (Ctrl) or Bim siRNA and analyzed as described above. The Bim Western blot depicts an extended molecular weight range to show that Bim-EL is the predominant form of the protein expressed in SET-2 cells. C: SET-2 cells were transfected with control, Bad or Bim siRNAs, or siRNAs to deplete both Bad and Bim simultaneously, for 48 hours and then treated with 1 μM NVP-BSK805 or the drug vehicle DMSO for 40 hours. Controls also consisted of non-transfected cells. Then, DNA content was measured by FACS using propidium iodide (PI) staining and the percentage of cells with less than 2N DNA content was determined (data represent the mean ± SD of three independent experiments). *Significantly different from respective DMSO control using t-test (p < 0.05). #Significant difference between groups as assessed by t-test (p < 0.05). D: SET-2 cells were transfected with control, Bad or Bim siRNAs, or siRNAs to deplete both Bad and Bim simultaneously, for 24 hours and then treated with increasing concentrations of NVP-BSK805 in cell proliferation assays for 48 hours. The histogram depicts half-maximal growth-inhibitory (GI50) concentrations of NVP-BSK805 for each condition (data represent the mean ± SD of two independent experiments carried out in triplicate).

Mentions: To gain more insights into the apoptotic players involved in triggering the caspases of the intrinsic pathway in JAK2V617F cell lines, we tested the impact of Bad depletion on JAK2 inhibitor-induced apoptosis. Bad and Bcl-xL have previously been shown to play a role in SET-2 cell survival [10]. In agreement with these earlier reports, Bad depletion by RNAi partially suppressed apoptosis induction in SET-2 cells, as assessed by PARP cleavage and measuring the sub-G1 cell fraction by flow cytometry, following JAK2 inhibition (Figure 3A and 3C). However, in MB-02 cells Bad depletion only modestly suppressed NVP-BSK805-induced cell death (Figure 4A and 4C). Intrigued by this finding, we explored the role of Bim, another BH3-only protein, in JAK2 inhibitor-induced apoptosis. In both cell lines, Bim levels were readily detected at baseline and strongly reduced following RNAi (Figure 2, 3A and 4A). In both SET-2 and MB02 cells Bim-EL was the predominant isoform expressed (Figure 3B and 4B). Importantly, Bim-depleted SET-2 and MB-02 cells were largely resistant to cell death by NVP-BSK805 (Figure 3C and 4C). Similarly, Will et al. recently reported that shRNA-mediated Bim depletion suppressed apoptosis induced by JAK2 inhibition in HEL cells [21]. In SET-2 cell proliferation assays, Bim depletion resulted in a three-fold increase in the GI50 (half-maximal growth-inhibitory concentration) of NVP-BSK805 (Figure 3D). In agreement with a recent report [21], these findings corroborate a crucial role for Bim in the execution of cell death in JAK2V617F mutant cells.


Bim and Mcl-1 exert key roles in regulating JAK2V617F cell survival.

Rubert J, Qian Z, Andraos R, Guthy DA, Radimerski T - BMC Cancer (2011)

Bad and Bim depletion in JAK2V617F mutant SET-2 cells suppress NVP-BSK805-induced apoptosis. A: SET-2 cells were transfected with control siRNA (Ctrl), Bad siRNA or siRNAs to deplete both Bad and Bim simultaneously. After 72 hours, cells were treated with 500 nM NVP-BSK805 or the drug vehicle DMSO for 30 hours and extracted for Western blot analysis. Arrowhead depicts cleaved PARP running below the full-length PARP protein. β-tubulin was probed for as a loading control. B: SET-2 cells were transfected with control siRNA (Ctrl) or Bim siRNA and analyzed as described above. The Bim Western blot depicts an extended molecular weight range to show that Bim-EL is the predominant form of the protein expressed in SET-2 cells. C: SET-2 cells were transfected with control, Bad or Bim siRNAs, or siRNAs to deplete both Bad and Bim simultaneously, for 48 hours and then treated with 1 μM NVP-BSK805 or the drug vehicle DMSO for 40 hours. Controls also consisted of non-transfected cells. Then, DNA content was measured by FACS using propidium iodide (PI) staining and the percentage of cells with less than 2N DNA content was determined (data represent the mean ± SD of three independent experiments). *Significantly different from respective DMSO control using t-test (p < 0.05). #Significant difference between groups as assessed by t-test (p < 0.05). D: SET-2 cells were transfected with control, Bad or Bim siRNAs, or siRNAs to deplete both Bad and Bim simultaneously, for 24 hours and then treated with increasing concentrations of NVP-BSK805 in cell proliferation assays for 48 hours. The histogram depicts half-maximal growth-inhibitory (GI50) concentrations of NVP-BSK805 for each condition (data represent the mean ± SD of two independent experiments carried out in triplicate).
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Figure 3: Bad and Bim depletion in JAK2V617F mutant SET-2 cells suppress NVP-BSK805-induced apoptosis. A: SET-2 cells were transfected with control siRNA (Ctrl), Bad siRNA or siRNAs to deplete both Bad and Bim simultaneously. After 72 hours, cells were treated with 500 nM NVP-BSK805 or the drug vehicle DMSO for 30 hours and extracted for Western blot analysis. Arrowhead depicts cleaved PARP running below the full-length PARP protein. β-tubulin was probed for as a loading control. B: SET-2 cells were transfected with control siRNA (Ctrl) or Bim siRNA and analyzed as described above. The Bim Western blot depicts an extended molecular weight range to show that Bim-EL is the predominant form of the protein expressed in SET-2 cells. C: SET-2 cells were transfected with control, Bad or Bim siRNAs, or siRNAs to deplete both Bad and Bim simultaneously, for 48 hours and then treated with 1 μM NVP-BSK805 or the drug vehicle DMSO for 40 hours. Controls also consisted of non-transfected cells. Then, DNA content was measured by FACS using propidium iodide (PI) staining and the percentage of cells with less than 2N DNA content was determined (data represent the mean ± SD of three independent experiments). *Significantly different from respective DMSO control using t-test (p < 0.05). #Significant difference between groups as assessed by t-test (p < 0.05). D: SET-2 cells were transfected with control, Bad or Bim siRNAs, or siRNAs to deplete both Bad and Bim simultaneously, for 24 hours and then treated with increasing concentrations of NVP-BSK805 in cell proliferation assays for 48 hours. The histogram depicts half-maximal growth-inhibitory (GI50) concentrations of NVP-BSK805 for each condition (data represent the mean ± SD of two independent experiments carried out in triplicate).
Mentions: To gain more insights into the apoptotic players involved in triggering the caspases of the intrinsic pathway in JAK2V617F cell lines, we tested the impact of Bad depletion on JAK2 inhibitor-induced apoptosis. Bad and Bcl-xL have previously been shown to play a role in SET-2 cell survival [10]. In agreement with these earlier reports, Bad depletion by RNAi partially suppressed apoptosis induction in SET-2 cells, as assessed by PARP cleavage and measuring the sub-G1 cell fraction by flow cytometry, following JAK2 inhibition (Figure 3A and 3C). However, in MB-02 cells Bad depletion only modestly suppressed NVP-BSK805-induced cell death (Figure 4A and 4C). Intrigued by this finding, we explored the role of Bim, another BH3-only protein, in JAK2 inhibitor-induced apoptosis. In both cell lines, Bim levels were readily detected at baseline and strongly reduced following RNAi (Figure 2, 3A and 4A). In both SET-2 and MB02 cells Bim-EL was the predominant isoform expressed (Figure 3B and 4B). Importantly, Bim-depleted SET-2 and MB-02 cells were largely resistant to cell death by NVP-BSK805 (Figure 3C and 4C). Similarly, Will et al. recently reported that shRNA-mediated Bim depletion suppressed apoptosis induced by JAK2 inhibition in HEL cells [21]. In SET-2 cell proliferation assays, Bim depletion resulted in a three-fold increase in the GI50 (half-maximal growth-inhibitory concentration) of NVP-BSK805 (Figure 3D). In agreement with a recent report [21], these findings corroborate a crucial role for Bim in the execution of cell death in JAK2V617F mutant cells.

Bottom Line: Treatment of JAK2V617F mutant cell lines with a JAK2 inhibitor was found to trigger Bim activation.We conclude that Bim and Mcl-1 have key opposing roles in regulating JAK2V617F cell survival and propose that inactivation of aberrant JAK2 signaling leads to changes in Bim complexes that trigger cell death.Thus, further preclinical evaluation of combinations of JAK2 inhibitors with Bcl-2 family antagonists that also tackle Mcl-1, besides Bcl-xL, is warranted to assess the therapeutic potential for the treatment of chronic myeloproliferative neoplasms.

View Article: PubMed Central - HTML - PubMed

Affiliation: Disease Area Oncology, Novartis Institutes for BioMedical Research, Basel, Switzerland.

ABSTRACT

Background: The JAK2V617F mutation plays a major role in the pathogenesis of myeloproliferative neoplasms and is found in the vast majority of patients suffering from polycythemia vera and in roughly every second patient suffering from essential thrombocythemia or from primary myelofibrosis. The V617F mutation is thought to provide hematopoietic stem cells and myeloid progenitors with a survival and proliferation advantage. It has previously been shown that activated JAK2 promotes cell survival by upregulating the anti-apoptotic STAT5 target gene Bcl-xL. In this study, we have investigated the role of additional apoptotic players, the pro-apoptotic protein Bim as well as the anti-apoptotic protein Mcl-1.

Methods: Pharmacological inhibition of JAK2/STAT5 signaling in JAK2V617F mutant SET-2 and MB-02 cells was used to study effects on signaling, cell proliferation and apoptosis by Western blot analysis, WST-1 proliferation assays and flow cytometry. Cells were transfected with siRNA oligos to deplete candidate pro- and anti-apoptotic proteins. Co-immunoprecipitation assays were performed to assess the impact of JAK2 inhibition on complexes of pro- and anti-apoptotic proteins.

Results: Treatment of JAK2V617F mutant cell lines with a JAK2 inhibitor was found to trigger Bim activation. Furthermore, Bim depletion by RNAi suppressed JAK2 inhibitor-induced cell death. Bim activation following JAK2 inhibition led to enhanced sequestration of Mcl-1, besides Bcl-xL. Importantly, Mcl-1 depletion by RNAi was sufficient to compromise JAK2V617F mutant cell viability and sensitized the cells to JAK2 inhibition.

Conclusions: We conclude that Bim and Mcl-1 have key opposing roles in regulating JAK2V617F cell survival and propose that inactivation of aberrant JAK2 signaling leads to changes in Bim complexes that trigger cell death. Thus, further preclinical evaluation of combinations of JAK2 inhibitors with Bcl-2 family antagonists that also tackle Mcl-1, besides Bcl-xL, is warranted to assess the therapeutic potential for the treatment of chronic myeloproliferative neoplasms.

Show MeSH
Related in: MedlinePlus